Fig 1: The transcriptional activity of mammalian IRF7 depends on covalent attachment of NEDD8 to its C-terminal lysines.(A) A schematic representation of all the lysine residues in murine IRF7. (B) HEK-293T cells were transfected with mammalian expression vectors encoding His-NEDD8 and FLAG-Myc-tagged murine IRF7 WT or mutants. Twenty-four hours later, the neddylation of exogenous murine IRF7 was examined by IB analysis with the indicated antibodies after histidine pulldown under fully denaturing conditions. (C) Bacterially expressed His-tagged murine IRF7 WT or mutants were subjected to in vitro neddylation (37°C, 1 h), followed by IB with the indicated antibodies. (D-G) FLAG-Myc-tagged murine IRF7 WT and mutants were co-transfected with reporter plasmids driven by Ifna4 promoter or Ifna6 promoter in HEK-293T cells. After 12 h, the cells were treated with 0.5µM MLN4924 for another 24 h or left untreated. Dual-reporter luciferase (LUC) assays were then performed with the supernatants. Luciferase activity was reported as fold induction (Top). Cell lysates were subjected to IB analysis with antibodies against Myc tag and ß-actin (Bottom). Quantitative data are shown as Mean ± SD (n = 3 ~ 4 per group). **p< 0.01; ***p< 0.001; NS, not significant.